Nuclear import is accomplished by nucleocytoplasmic shuttling receptors (importins) that transport cargo though the nuclear pore complex (NPC) by interaction with a series of nucleoporins. One of the critical regulators of import is the small GTPase Ran. The long-term goal of this work is to contribute to a detailed understanding of nucleocytoplasmic transport. This project is focused on molecularly characterizing how import complexes move through the NPC, and how this process is regulated by Ran. The specific aims are: 1) The role of RanGTP in facilitating large cargo transport through the NPC will be analyzed in permeabilized cell assays using importin beta mutants deficient in nucleoporin binding and cargoes of varying size and import signal density. 2) The structure of a complex of RanGTP with full-length importin beta will be determined by X-ray crystallography. Importin beta mutants affecting Ran binding that are predicted to be deficient in cargo release will be examined. 3) The properties of a snurportin IBB/importin beta complex will be analyzed by biochemical and functional approaches. 4) The possibility that other import receptor complexes besides importin alpha/beta have a gradient of increasing affinity from the cytoplasmic-to-nuclear side of the NPC will be examined, and the functions of this gradient will be tested in permeabilized cell assays. The role of a nuclear-to-cytoplasmic nucleoporin affinity gradient in the recycling of importin beta/RanGTP complexes will be analyzed. It is hoped that this work will shed new light on some fundamental aspects of nuclear import mechanisms. Nucleocytoplasmic transport is central to the functioning of eukaryotic cells, and is an integral part of the processes that lead to most human diseases. Thus, understanding basic nuclear transport mechanisms can provide a framework for developing new therapeutic approaches relevant to viral pathogenesis, cancer and immunity and other processes.